RESUMEN
Apart from the canonical serotonin (5-hydroxytryptamine [5-HT])-receptor signaling transduction pattern, 5-HT-involved post-translational serotonylation has recently been noted. Here, we report a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serotonylation system that promotes the glycolytic metabolism and antitumor immune activity of CD8+ T cells. Tissue transglutaminase 2 (TGM2) transfers 5-HT to GAPDH glutamine 262 and catalyzes the serotonylation reaction. Serotonylation supports the cytoplasmic localization of GAPDH, which induces a glycolytic metabolic shift in CD8+ T cells and contributes to antitumor immunity. CD8+ T cells accumulate intracellular 5-HT for serotonylation through both synthesis by tryptophan hydroxylase 1 (TPH1) and uptake from the extracellular compartment via serotonin transporter (SERT). Monoamine oxidase A (MAOA) degrades 5-HT and acts as an intrinsic negative regulator of CD8+ T cells. The adoptive transfer of 5-HT-producing TPH1-overexpressing chimeric antigen receptor T (CAR-T) cells induced a robust antitumor response. Our findings expand the known range of neuroimmune interaction patterns by providing evidence of receptor-independent serotonylation post-translational modification.
Asunto(s)
Linfocitos T CD8-positivos , Serotonina , Linfocitos T CD8-positivos/metabolismo , Serotonina/metabolismo , Serotonina/farmacología , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH.
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Glucosa , Hexoquinasa/metabolismo , Animales , Glucosa/metabolismo , Glucólisis , Hexoquinasa/genética , Ratones , Mitocondrias/metabolismo , Vía de Pentosa FosfatoRESUMEN
The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.
Asunto(s)
Citosol/metabolismo , Glutamina/metabolismo , Malato Deshidrogenasa/metabolismo , Mitocondrias/patología , NAD/metabolismo , Osteosarcoma/patología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Movimiento Celular , Ciclo del Ácido Cítrico , ADN Mitocondrial/genética , Metabolismo Energético , Femenino , Glucosa/metabolismo , Glucólisis , Humanos , Mitocondrias/metabolismo , Osteosarcoma/genética , Osteosarcoma/metabolismo , Oxidación-Reducción , Células Tumorales CultivadasRESUMEN
Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.
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Lisosomas/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/genética , NAD/metabolismo , Animales , Células Cultivadas , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/deficiencia , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Fosfoglicerato Quinasa/metabolismoRESUMEN
How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.
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Arabidopsis/efectos de los fármacos , Dipéptidos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/antagonistas & inhibidores , Nicotiana/efectos de los fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Simulación por Computador , Dipéptidos/química , Dipéptidos/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , NADP/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacos , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Proteínas de Plantas/antagonistas & inhibidores , Plantones/efectos de los fármacos , Plantones/metabolismo , Nicotiana/metabolismoRESUMEN
Small noncoding RNAs fulfill key functions in cellular and organismal biology, typically working in concert with RNA-binding proteins (RBPs). While proteome-wide methodologies have enormously expanded the repertoire of known RBPs, these methods do not distinguish RBPs binding to small noncoding RNAs from the rest. To specifically identify this relevant subclass of RBPs, we developed small noncoding RNA interactome capture (snRIC2C) based on the differential RNA-binding capacity of silica matrices (2C). We define the S. cerevisiae proteome of nearly 300 proteins that specifically binds to RNAs smaller than 200 nt in length (snRBPs), identifying informative distinctions from the total RNA-binding proteome determined in parallel. Strikingly, the snRBPs include most glycolytic enzymes from yeast. With further methodological developments using silica matrices, 12 tRNAs were identified as specific binders of the glycolytic enzyme GAPDH. We show that tRNA engagement of GAPDH is carbon source-dependent and regulated by the RNA polymerase III repressor Maf1, suggesting a regulatory interaction between glycolysis and RNA polymerase III activity. We conclude that snRIC2C and other 2C-derived methods greatly facilitate the study of RBPs, revealing previously unrecognized interactions.
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Glucólisis , ARN Pequeño no Traducido , ARN de Transferencia , Proteínas de Unión al ARN , Saccharomyces cerevisiae , Glucólisis/genética , Proteoma/genética , ARN/metabolismo , ARN Polimerasa III/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismoRESUMEN
Homologous recombination (HR), a form of error-free DNA double-strand break (DSB) repair, is important for the maintenance of genomic integrity. Here, we identify a moonlighting protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a regulator of HR repair, which is mediated through HDAC1-dependent regulation of RAD51 stability. Mechanistically, in response to DSBs, Src signaling is activated and mediates GAPDH nuclear translocation. Then, GAPDH directly binds with HDAC1, releasing it from its suppressor. Subsequently, activated HDAC1 deacetylates RAD51 and prevents it from undergoing proteasomal degradation. GAPDH knockdown decreases RAD51 protein levels and inhibits HR, which is re-established by overexpression of HDAC1 but not SIRT1. Notably, K40 is an important acetylation site of RAD51, which facilitates stability maintenance. Collectively, our findings provide new insights into the importance of GAPDH in HR repair, in addition to its glycolytic activity, and they show that GAPDH stabilizes RAD51 by interacting with HDAC1 and promoting HDAC1 deacetylation of RAD51.
Asunto(s)
Reparación del ADN , Reparación del ADN por Recombinación , Recombinación Homóloga , Roturas del ADN de Doble Cadena , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismoRESUMEN
Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90â¢apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.
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Gliceraldehído-3-Fosfato Deshidrogenasas , Proteínas HSP70 de Choque Térmico , Proteínas HSP90 de Choque Térmico , Hemo , Hemoproteínas , Chaperonas Moleculares , Óxido Nítrico Sintasa , Hemo/química , Hemoproteínas/química , Hemoproteínas/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/metabolismo , Enzimas Inmovilizadas , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Activación EnzimáticaRESUMEN
Indoleamine-2, 3-dioxygenase (IDO1) and Tryptophan-2, 3-dioxygenase (TDO) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and thus play primary roles in metabolism, inflammation, and tumor immune surveillance. Because their activities depend on their heme contents, which vary in biological settings and go up or down in a dynamic manner, we studied how their heme levels may be impacted by nitric oxide (NO) in mammalian cells. We utilized cells expressing TDO or IDO1 either naturally or via transfection and determined their activities, heme contents, and expression levels as a function of NO exposure. We found NO has a bimodal effect: a narrow range of low NO exposure promoted cells to allocate heme into the heme-free TDO and IDO1 populations and consequently boosted their heme contents and activities 4- to 6-fold, while beyond this range the NO exposure transitioned to have a negative impact on their heme contents and activities. NO did not alter dioxygenase protein expression levels, and its bimodal impact was observed when NO was released by a chemical donor or was generated naturally by immune-stimulated macrophage cells. NO-driven heme allocations to IDO1 and TDO required participation of a GAPDH-heme complex and for IDO1 required chaperone Hsp90 activity. Thus, cells can up- or downregulate their IDO1 and TDO activities through a bimodal control of heme allocation by NO. This mechanism has important biomedical implications and helps explain why the IDO1 and TDO activities in animals go up and down in response to immune stimulation.
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Indolamina-Pirrol 2,3,-Dioxigenasa , Óxido Nítrico , Triptófano Oxigenasa , Animales , Hemo/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Mamíferos/metabolismo , Triptófano/metabolismo , Triptófano Oxigenasa/química , Triptófano Oxigenasa/metabolismoRESUMEN
Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.
Asunto(s)
Vías Biosintéticas , Ciclina D1 , Hepatocitos , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Hepatocitos/metabolismo , Proteómica , Pirimidinas/biosíntesis , Humanos , Animales , Ratones , Línea CelularRESUMEN
A "switch" in the metabolic pattern of microglia is considered to be required to meet the metabolic demands of cell survival and functions. However, how metabolic switches regulate microglial function remains controversial. We found here that exposure to amyloid-ß triggers microglial inflammation accompanied by increasing GAPDH levels. The increase of GAPDH, a glycolysis enzyme, leads to the reduced release of interferon-γ (IFN-γ) from inflammatory microglia. Such alternation is translational and is regulated by the binding of glycolysis enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to IFN-γ mRNA. GAPDH, by engaging/disengaging glycolysis and through influencing IFN-γ expression, regulates microglia functions, including phagocytosis and cytokine production. Phosphoglycerate dehydrogenase (PHGDH), screened from different state microglia by metabolomics combined with METARECON analysis, is a metabolic enzyme adjacent downstream of GAPDH and synthesizes serine on the collateral pathway derived from glycolysis. Polarization of microglial with PHGDH as a metabolic checkpoint can be bidirectionally regulated by adding IL-4 or giving PHGDH inhibitors. Therefore, regulation of metabolic enzymes not only reprograms metabolic patterns, but also manipulates microglia functions. Further study should be performed to explore the mechanism of metabolic checkpoints in human microglia or more in vivo animal experiments, and may expand to the effects of various metabolic substrates or enzyme, such as lipids and amino acids, on the functions of microglia.
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Microglía , Fosfoglicerato-Deshidrogenasa , Animales , Humanos , Fosfoglicerato-Deshidrogenasa/genética , Interferón gamma , Multiómica , Gliceraldehído-3-Fosfato Deshidrogenasas/genéticaRESUMEN
Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts.
RESUMEN
The Golgi complex is a highly dynamic organelle that regulates various cellular activities and yet maintains a distinct structure. Multiple proteins participate in Golgi structure/organization including the small GTPase Rab2. Rab2 is found on the cis/medial Golgi compartments and the endoplasmic reticulum-Golgi intermediate compartment. Interestingly, Rab2 gene amplification occurs in a wide range of human cancers and Golgi morphological alterations are associated with cellular transformation. To learn how Rab2 'gain of function' influences the structure/activity of membrane compartments in the early secretory pathway that may contribute to oncogenesis, NRK cells were transfected with Rab2B cDNA. We found that Rab2B overexpression had a dramatic effect on the morphology of pre- and early Golgi compartments that resulted in a decreased transport rate of VSV-G in the early secretory pathway. We monitored the cells for the autophagic marker protein LC3 based on the findings that depressed membrane trafficking affects homeostasis. Morphological and biochemical studies confirmed that Rab2 ectopic expression stimulated LC3-lipidation on Rab2-containing membranes that was dependent on GAPDH and utilized a non-canonical LC3-conjugation mechanism that is nondegradative. Golgi structural alterations are associated with changes in Golgi-associated signalling pathways. Indeed, Rab2 overexpressing cells had elevated Src activity. We propose that increased Rab2 expression facilitates cis Golgi structural changes that are maintained and tolerated by the cell due to LC3 tagging, and subsequent membrane remodeling triggers Golgi associated signaling pathways that may contribute to oncogenesis.
Asunto(s)
Aparato de Golgi , Orgánulos , Humanos , Aparato de Golgi/metabolismo , Orgánulos/metabolismo , Transporte Biológico , Autofagia , Carcinogénesis/metabolismoRESUMEN
Obtaining accurate and reliable gene expression results in real-time RT-PCR (qRT-PCR) data analysis requires appropriate normalization by carefully selected reference genes, either a single or a combination of multiple housekeeping genes (HKGs). The optimal reference gene/s for normalization should demonstrate stable expression across varying conditions to diminish potential influences on the results. Despite the extensive database available, research data are lacking regarding the most appropriate HKGs for qRT-PCR data analysis in rabbit and horse adipose-derived stem cells (ASCs). Therefore, in our study, we comprehensively assessed and compared the suitability of some widely used HKGs, employing RefFinder and NormFinder, two extensively acknowledged algorithms for robust data interpretation. The rabbit and horse ASCs were obtained from subcutaneous stromal vascular fraction. ASCs were induced into tri-lineage differentiation, followed by the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) treatment of the adipose-differentiated rabbit ASCs, while horse experimental groups were formed based on adipogenic, osteogenic, and chondrogenic differentiation. At the end of the experiment, the total mRNA was obtained and used for the gene expression evaluation of the observed factors. According to our findings, glyceraldehyde 3-phosphate dehydrogenase was identified as the most appropriate endogenous control gene for rabbit ASCs, while hypoxanthine phosphoribosyltransferase was deemed most suitable for horse ASCs. The obtained results underscore that these housekeeping genes exhibit robust stability across diverse experimental conditions, remaining unaltered by the treatments. In conclusion, the current research can serve as a valuable baseline reference for experiments evaluating gene expression in rabbit and horse ASCs. It highlights the critical consideration of housekeeping gene abundance and stability in qPCR experiments, emphasizing the need for an individualized approach tailored to the specific requirements of the study.
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Genes Esenciales , Gliceraldehído-3-Fosfato Deshidrogenasas , Caballos , Conejos , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Diferenciación Celular , Adipogénesis , Estándares de Referencia , Perfilación de la Expresión Génica/métodosRESUMEN
Cell cycle progression and division is regulated by checkpoint controls and sequential activation of cyclin-dependent kinases (CDKs). Understanding of how these events occur in synchrony with metabolic changes could have important therapeutic implications. For biosynthesis, cancer cells enhance glucose and glutamine consumption. Inactivation of pyruvate kinase M2 (PKM2) promotes transcription in G1 phase. Glutamine metabolism supports DNA replication in S phase and lipid synthesis in G2 phase. A boost in glycolysis and oxidative metabolism can temporarily furnish more ATP when necessary (G1/S transition, segregation of chromosomes). Recent studies have shown that a few metabolic enzymes [PKM2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3), GAPDH] also periodically translocate to the nucleus and oversee cell cycle regulators or oncogene expression (c-Myc). Targeting these metabolic enzymes could increase the response to CDK inhibitors (CKIs).
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Ciclo Celular , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Quinasas/metabolismo , Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Autophagy is considered to be an important switch for facilitating normal to malignant cell transformation during colorectal cancer development. Consistent with other reports, we found that the membrane receptor Neuropilin1 (NRP1) is greatly upregulated in colon cancer cells that underwent autophagy upon glucose deprivation. However, the mechanism underlying NRP1 regulation of autophagy is unknown. We found that knockdown of NRP1 inhibits autophagy and largely upregulates the expression of aldo-keto reductase family 1 B10 (AKR1B10). Moreover, we demonstrated that AKR1B10 interacts with and inhibits the nuclear importation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and then subsequently represses autophagy. Interestingly, we also found that an NADPH-dependent reduction reaction could be induced when AKR1B10 interacts with GAPDH, and the reductase activity of AKR1B10 is important for its repression of autophagy. Together, our findings unravel a novel mechanism of NRP1 in regulating autophagy through AKR1B10.
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Aldehído Reductasa , Neoplasias del Colon , Aldehído Reductasa/genética , Aldo-Ceto Reductasas , Autofagia , Neoplasias del Colon/genética , Glucosa , Gliceraldehído-3-Fosfato Deshidrogenasas , HumanosRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acts as a sensor under oxidative stress, leading to induction of various biological responses. Given that mitogen-activated protein kinase (MAPK) signaling pathways mediate cellular responses to a wide variety of stimuli, including oxidative stress, here, we aimed to elucidate whether a cross-talk cascade between GAPDH and MAPKs occurs under oxidative stress. Of the three typical MAPKs investigated-extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase (JNK)-we found that hydrogen peroxide (H2O2)-induced JNK activation is significantly reduced in HEK293 cells treated with small-interfering (si)RNA targeting GAPDH. Co-immunoprecipitation with a GAPDH antibody further revealed protein-protein interactions between GAPDH and JNK in H2O2-stmulated cells. Notably, both JNK activation and these interactions depend on oxidation of the active-site cysteine (Cys152) in GAPDH, as demonstrated by rescue experiments with either exogenous wild-type GAPDH or the cysteine-substituted mutant (C152A) in endogenous GAPDH-knockdown HEK293 cells. Moreover, H2O2-induced translocation of Bcl-2-associated X protein (Bax) into mitochondria, which occurs downstream of JNK activation, is attenuated by endogenous GAPDH knockdown in HEK293 cells. These results suggest a novel role for GAPDH in the JNK signaling pathway under oxidative stress.
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Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante) , Peróxido de Hidrógeno , Proteínas Quinasas JNK Activadas por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Humanos , Cisteína/metabolismo , Células HEK293 , Peróxido de Hidrógeno/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Estrés Oxidativo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismoRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be one of the targets of methylglyoxal (MGO), a metabolite of glycolysis that increased in diabetes. However, the mechanism of GAPDH inactivation in the presence of MGO is unclear. The purpose of the work was to study the reaction of GAPDH with MGO and to identify the products of the reaction. It was shown that incubation of recombinant human GAPDH with MGO leads to irreversible inactivation of the enzyme, which is accompanied by a decrease in SH-group content by approximately 3.3 per tetramer GAPDH. MALDI-TOF MS analysis showed that the modification of GAPDH with MGO results in the oxidation of the catalytic cysteine residues (Cys152) to form cysteine-sulfinic acid. In addition, 2 arginine residues (R80 and R234) were identified that react with MGO to form hydroimidazolones. Incubation of SH-SY5Y neuroblastoma cells with MGO resulted in the inactivation of GAPDH and inhibition of glycolysis. The mechanism of GAPDH oxidation in the presence of MGO suggests the participation of superoxide anion, which is formed during the reaction of amino groups with methylglyoxal. The role of GAPDH in protection against the damaging effect of ROS in cells in the case of inefficiency of MGO removal by the GSH-dependent glyoxalase system is discussed.
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Neuroblastoma , Piruvaldehído , Humanos , Cisteína/metabolismo , Óxido de Magnesio , Gliceraldehído-3-Fosfato Deshidrogenasas/químicaRESUMEN
GAPDH, a heme chaperone, has been previously implicated in the incorporation of heme into iNOS and soluble guanylyl cyclase (sGC). Since sGC is critical for myoglobin (Mb) heme-maturation, we investigated the role of GAPDH in the maturation of this globin, as well as hemoglobins α, ß, and γ. Utilizing cell culture systems, we found that overexpression of wild-type GAPDH increased, whereas GAPDH mutants H53A and K227A decreased, the heme content of Mb and Hbα and Hbß. Overexpression of wild-type GAPDH fully recovered the heme-maturation inhibition observed with the GAPDH mutants. Partial rescue was observed by overexpression of sGCß1 but not by overexpression of a sGCΔß1 deletion mutant, which is unable to bind the sGCα1 subunit required to form the active sGCα1ß1 complex. Wild type and mutant GAPDH was found to be associated in a complex with each of the globins and Hsp90. GAPDH at endogenous levels was found to be associated with Mb in differentiating C2C12 myoblasts, and with Hbγ or Hbα in differentiating HiDEP-1 erythroid progenitor cells. Knockdown of GAPDH in C2C12 cells suppressed Mb heme-maturation. GAPDH knockdown in K562 erythroleukemia cells suppressed Hbα and Hbγ heme-maturation as well as Hb dimerization. Globin heme incorporation was not only dependent on elevated sGCα1ß1 heterodimer formation, but also influenced by iron provision and magnitude of expression of GAPDH, d-aminolevulinic acid, and FLVCR1b. Together, our data support an important role for GAPDH in the maturation of myoglobin and γ, ß, and α hemoglobins.
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Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Hemo/metabolismo , Hemoglobinas/metabolismo , Chaperonas Moleculares/metabolismo , Mioglobina/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Células HEK293 , Hemo/genética , Hemoglobinas/genética , Humanos , Células K562 , Chaperonas Moleculares/genética , Mutación Missense , Mioglobina/genética , Sarcoglicanos/genética , Sarcoglicanos/metabolismoRESUMEN
BACKGROUND: Maconellicoccus hirsutus is a destructive pest which causes severe losses of agricultural and horticultural crops. For the management of M. hirsutus, many insecticides have been used and it has been exposed to insufficient dosage or uneven spray coverage which resulted in the development of insecticide resistance. Xenobiotic metabolism can be better understood with the help of gene expression studies by unveiling the underlying molecular mechanisms. The qRT-PCR is the simplest method to analyse gene expression, however, it highly relies on suitable reference genes concerning the different experimental conditions. METHODS AND RESULTS: We evaluated the stability of five reference genes in two sets of experimental conditions viz. developmental stages (nymphs and adults) and agrochemical stress (GA3 and Buprofezin sprayed) against M. hirsutus, using different softwares-NormFinder, geNorm, BestKeeper, and RefFinder. The study revealed that ATP51a and GAPDH can be used as reference genes for gene expression studies when exposed to Gibberellic acid. Additionally, the study revealed that the ideal pair of reference genes for data validation in M. hirsutus treated with Buprofezin was GAPDH and ß-tubulin. The ideal reference gene combination for various developmental stages was found to be 28S and Actin. CONCLUSION: According to the study, GAPDH can be utilized as a reliable reference gene in the agrochemical (GA3 and Buprofezin) exposure set. The genes can be utilized as a suitable reference for qRT-PCR gene expression studies of xenobiotic metabolism to understand the underlying molecular mechanism, which will help further to design suitable management strategies.